Site specific radioiodination of recombinant hirudin.
Recombinant hirudin variant rHV2-Lys47 was radioiodinated using the chloramine-T method. Depending on the reaction pH, the two tyrosine residues, Tyr3 and Tyr63, responded differently to iodination but without change in total iodination yield. Of the incorporated -125 iodine 80% was located on Tyr3 at pH 7.4, but 65% was found on Tyr63 at pH 4. These distinct iodination patterns suggest the existence of a pH-dependent multimerization and/or important conformational changes in the tertiary structure with pH. Each radiotracer was purified to high specific activity by simple low-pressure chromatography including gel filtration and reverse-phase separation, both on short cartridges. The method was validated by reverse-phase and anion-exchange HPLC with on-line radioactivity detection. The iodination sites were characterized following carboxypeptidase Y cleavage coupled with radio-HPLC.
Tuong
A
A
Service d'Analyse de Recherche, Sanofi Recherche, Toulouse, France.
Maftouh
M
M
Picard
C
C
Gachon
M
M
D000595:Amino Acid Sequence; D002268:Carboxypeptidases / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange; D014644:Genetic Variation; D006629:Hirudins / Q000737:chemistry* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007455:Iodine; D007457:Iodine Radioisotopes / Q000737:chemistry; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D006629:Hirudins; D007457:Iodine Radioisotopes; D011994:Recombinant Proteins; D007455:Iodine; D002268:Carboxypeptidases; C022633:serine carboxypeptidase